Method of collecting placental stem cells

ABSTRACT

A method of collecting embryonic-like stem cells from a placenta which has been treated to remove residual cord blood by perfusing the drained placenta with an anticoagulant solution to flush out residual cells, collecting the residual cells and perfusion liquid from the drained placenta, and separating the embryonic-like cells from the residual cells and perfusion liquid. Exogenous cells can be propagated in the placental bioreactor and bioactive molecules collected therefrom.

BENEFIT OF PRIOR PROVISIONAL APPLICATION

This application is a divisional of U.S. application Ser. No.12/618,664, filed Nov. 13, 2009, which is a continuation of U.S.application Ser. No. 11/187,400 (now U.S. Pat. No. 7,638,141), filedJul. 21, 2005, which is a continuation of U.S. application Ser. No.10/004,942 (now U.S. Pat. No. 7,045,148), filed Dec. 5, 2001, whichclaims benefit of U.S. Provisional Application No. 60/251,900, filedDec. 6, 2000, the specifications of which are each incorporated hereinby reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is generally in the area of stem cell collection,and particularly in the recovery of embryonic-like stem cells and othermultipotent stem cells from placentas. These embryonic-like stem cellsare derived from the placenta collected after birth. Theseembryonic-like stem cells have characteristics of embryonic stem cellsbut are not derived from the embryo.

2. Description of the Background Art

Human stem cells are totipotential or pluripotential precursor cellscapable of generating a variety of mature human cell lineages. Thisability serves as the basis for the cellular differentiation andspecialization necessary for organ and tissue development. Recentsuccess at transplanting such stem cells have provided new clinicaltools to reconstitute and/or supplement the bone marrow aftermyeloablation due to disease, exposure to toxic chemical or radiation.Further evidence exists which demonstrates that stem cells can beemployed to repopulate many, if not all, tissues and restore physiologicand anatomic functionality. The application of stem cells in tissueengineering, gene therapy delivery and cell therapeutics is alsoadvancing rapidly.

Obtaining sufficient human stem cells has been problematic for severalreasons. First, isolation of normally occurring populations of stemcells in adult tissues has been technically difficult, costly and verylimited in quantity. Secondly, procurement of these cells from embryosor fetal tissue including abortuses has raised many ethical and moralconcerns. The widely held belief that the human embryo and fetusconstitute independent life has justified a moratorium on the use ofsuch sources for any purpose. Alternative sources which do not violatethe sanctity of independent life would be essential for further progressin the use of stem cells clinically.

Umbilical cord blood (cord blood) is a known source of hemopoieticpluripotent, progenitor stem cells that are cryopreserved for use inhemopoietic reconstitution. The use of cord blood for this purpose iswell known and is becoming a widely used therapeutic procedure. Theconventional technique for the collection of cord blood is based on theuse of a needle or cannula which is used with the aid of gravity todrain the cord blood from the placenta. Usually the needle or cannula isplaced in the umbilical vein and the placenta is gently massaged to aidin draining the cord blood from the placenta. Thereafter the drainedplacenta has been considered to be of no use and has typically beendiscarded. A major limitation of stem cell procurement from cord bloodhas been the frequently inadequate volume of cord blood obtainedresulting in insufficient cell numbers to reconstitute bone marrow aftertransplantation.

Stem cells are in critically short supply. These are important for thetreatment of a wide variety of disorders, including malignancies, inbornerrors of metabolism, hemoglobinopathies, and immunodeficiences. Itwould be highly advantageous to have a source of more embryonic stemcells.

Accordingly, it is a primary object of the present invention to providea method of extracting and recovering hematopoietic stem cells from anexsanguinated placenta.

It is also an object of the invention to provide a method for isolatingother embryonic-like and/or omnipotent stem cells from an extractant ofa drained placenta.

It is a further object of the invention to provide a method to collectstem cells from the umbilical cord vein, the best source of hemopoieticpluripotent progenitor stem cells.

It is a further object of the present invention to provide a method andmeans whereby additional embryonic-like stem cells in higherconcentrations can be obtained from a drained placenta.

It is a further object of the invention to provide a method of utilizingthe isolated and perfused placenta as a biorector providing a goodenvironment for the propagation of endogenous cells, including but notlimited to lymphocytes and stem cells.

It is a further object of the present invention to provide a method andmeans whereby stem cells can be obtained many hours following the birthand expulsion of the placenta from the uterus.

SUMMARY OF THE INVENTION

A method of extracting embryonic-like stem cells from a drained placentaby means of a perfusion technique which utilizes either or both of theumbilical artery and umbilical vein has been developed, on the recoveryof human placenta following exsanguination and collection of theresidual blood. The placenta is then processed in such a manner as toestablish an ex vivo, natural bioreactor environment in which residentstem cells within the parenchyma and extravascular space are recruitedand migrate into the empty microcirculation where they can be washedinto a collecting vessel by perfusion.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cross-sectional view of the cannulation of the vein andartery of a placenta to perfuse the placenta and then collect theperfusate.

FIGS. 2a-e are schematics showing the collection, clamping, perfusion,collection and storage of a drained and perfused placenta.

FIG. 3 is a cross-sectional schematic of a perfused placenta in a devicefor use as a bioreactor.

FIG. 4 is a selection scheme for sorting cells retrieved from a perfusedplacenta.

DETAILED DESCRIPTION OF THE INVENTION

I. Method of Draining and Extracting Placenta

Draining of Cord Blood and Storage of Fresh Placenta

The method requires access to freshly drained human placentas which havebeen subjected to a conventional cord blood recovery process by drainingsubstantially all of the cord blood from the placenta. It is importantthat the placenta be properly stored and drained if it is to be asuitable source of embryonic stem cells. Generally, a placenta should bestored in an anticoagulant solution at a temperature of 5 to 25° C.(centigrade) for no more than 48 hours prior to the collection of thecord blood. Suitable anticoagulant solutions are well known. A preferredanticoagulant solution comprises a solution of heparin (1% w/w in 1:1000solution). Generally, the drained placenta should be stored for no morethan 36 hours before the embryonic-like stem cells are collected.

Extraction of Cells

The preferred embryonic-like stem cell extraction method is based on theperfusion of the drained placenta with a suitable aqueous fluid such asan anticoagulant dissolved in any suitable aqueous isotonic fluid suchas 0.9N sodium chloride solution. The anticoagulant for the perfusionliquid may comprise heparin or warfarin sodium at a concentration whichis sufficient to prevent the formation of clots of any residual cordblood. Generally from 100 to 1000 units of heparin may be employed.

The extraction procedure is based on the passage of the perfusion liquidthrough either or both of the umbilical artery and umbilical vein usinga gravity flow into the placenta which is suspended in such a mannerthat the umbilical artery and umbilical vein are at the highest point.It is preferred to connect the umbilical artery and the umbilical veinsimultaneously as shown in FIG. 1 to a pipette that is connected via aflexible connector to a reservoir of the perfusion liquid which ispassed into the umbilical vein and artery and collected in a suitableopen vessel from the surface of the placenta that was attached to theuterus of the mother during gestation.

The collection technique is based on the use of a sufficient amount ofthe perfusion liquid that will result in the collection of the cellsleft after the drainage of the cord blood. It has been observed thatwhen the perfusion liquid is first collected, the liquid is colored withthe residual red blood cells and tends to become clear as the perfusionliquid is passed through the placenta. Generally from 30 to 100 ml ofperfusion liquid is adequate to collect the embryonic-like cells butmore or less may be used depending on the observed results.

II. Method of Using the Drained and Perfused Placenta as a BioReactor

Perfusion of the Drained Placenta

As discussed above, the placenta is recovered under aseptic conditionsfollowing exsanguination and clamping of the proximal umbilical cord(within 4-5 cm (centimeter) of the insertion into the placental disc)and placental blood recovery and transported in a sterile, thermallyinsulated (maintaining the temperature of the placenta between 20-28°C.) transport device to the laboratory for processing, for example, byplacing the clamped placenta in a sterile zip-lock plastic bag which isthen placed in an insulated Styrofoam container or vacuum insulationcontainer, as shown in FIGS. 2a -e.

Use of the Placenta as a Bioreactor

The placenta is placed in a sterile basin and washed with 500 mL ofphosphate-buffered normal saline. The wash fluid is discarded. Theumbilical vein is cannulated with a Teflon or plastic cannula connectedto sterile tubing which is connected to the perfusion manifold, as shownin FIG. 3. The basin is then covered and the placenta is maintained atroom temperature (20-25° C.) for a period varying from 2 to 24 hours.The placenta is then perfused at periodically, preferably at 4, 8, 12,and 24 hours, with a volume of perfusate, preferably 100 mL of perfusate(sterile normal saline supplemented with or without 1000 u/L heparinand/or EDTA and/or CPDA (creatine phosphate dextrose)). The effluentfluid which escapes the placenta at the opposite surface is collectedand processed to isolate the stem cells of interest. Alterations in theconditions in which the placenta is maintained and the nature of theperfusate can be made to modulate the volume and composition of theeffluent.

Stem cells are then isolated from the effluent using techniques known bythose skilled in the art, such as for example, density gradientcentrifugation, magnet cell separation or other acceptable method, andsorted, for example, according to the scheme shown in FIG. 4.

In variations of this method, the cells in the placenta can bestimulated to produce bioactive molecules, such as immunoglobulin, orother molecules, or stimulated to proliferate, for example, byadministration of erythropoietin. The cells can also be geneticallyengineered prior to harvest, while still in the bioreactor, or at thetime of harvest, using for example a viral vector such as an adenoviralor retroviral vector, or using mechanical means such as liposomal orchemical mediated uptake of the DNA.

The procedure is as follows:

-   1. Fully exsanguinate the placenta and remove any adherent    coagulated and non-adherent cellular contaminants.-   2. Cultivate and perfuse the placenta with perfusate solution (for    example, normal saline solution) with or without an anticoagulant,    and/or with or without an antimicrobial agent.-   3. Collect both the extravasated perfusate and circulated perfusate    into a sterile receptacle.-   4. Isolate cell types from the collected perfusate by employing    techniques known by those skilled in the art, such as for example,    but not limited to density gradient centrifugation, magnet cell    separation, affinity cell separation or differential adhesion    techniques.

In one embodiment of the invention, the placenta is used as a bioreactorfor endogenous cells, including but not limited to lymphocytes andvarious kinds of pluripotent and/or totipotent stem cells, by incubatingthe placenta for 48 hours with perfusate solution.

In another embodiment the placenta is processed to remove all endogenouscells and allowing foreign cells to be introduced and propagated in theenvironment of the perfused placenta. In a particular embodiment theperfused placenta is irradiated with electromagnetic, UV, X-ray, gamma-or beta-radiation to eradicate all remaining viable endogenous cells.The foreign cells of interest to be propagated in the irradiatedplacental bioreactor are then introduced.

The exogenous cells are induced to propagate by introduction ofnutrients and growth factors into the perfusion solution. Serum andother growth factors are added to the propagation perfusion solution ormedium. Growth factors are usually proteins and include but are notlimited to cytokines, lymphokines, interferons, colony stimulatingfactors (CSF's), interferons, chemokines, and interleukins. Other growthfactors include recombinant human hemopoietic growth factors includingligands, stem cell factors, thrombopoeitin (Tpo), interleukins, andgranulocyte colony-stimulating factor (G-CSF). The growth factorsintroduced into the perfusion solution can stimulate the propagation ofundifferentiated stems cells or differentiated hemopoietic cells andstimulate the production of bioactive molecules including but notlimited to immunoglobulins, hormones, or other growth factors aspreviously described.

The present invention will be further understood by reference to thefollowing example.

EXAMPLE 1 Perfusion of Drained Placenta

Twenty ml (milliliter) of phosphate buffered saline solution (PBS) isadded to the perfusion liquid and a 10 ml portion is collected andcentrifuged for 25 minutes at 3000 rpm (revolutions per minute). Theeffluent is divided into four tubes and placed in an ice bath. 2.5 ml ofa 1% fetal calf serum (FCS) solution in PBS is added and the tubes arecentrifuged (140 minutes×10 g (acceleration due to gravity)). The pelletis resuspended in 5 ml of 1% FCS and two tubes are combined. The totalmononucleocytes are calculated by adding the total lymphocytes and thetotal monocytes and multiplying this by the total cell suspensionvolume.

EXAMPLE 2 Analysis of Cells Obtained by Perfusion and Incubation ofPlacenta

WBC MID Total # of 1000/m1 Lym % % GRA % Volume Cells CB (Cord 10.5 43.28 48.8 60 ml 6.3 × 10⁸ Blood) PP (Placenta 12.0 62.9 18.2 18.9 15 ml 1.8× 10⁸ perfusate, room temperature) PP² 11.7 56.0 19.2 24.8 30 ml 3.5 ×10⁸ (Placenta perfusate, 37° C.) Samples of PP were after Ficoll. Totalcell number of PP after Ficoll is 5.3 × 10⁸ and number of CB beforeprocessing is 6.3 × 10⁸. Lym % is percent of lymphocytes; MID % ispercent of midrange white blood cells; and GRA % is percent ofgranulocytes.

Cell isolation is achieved by using magnetic cell separation, such asfor example, Auto Macs (Miltenyi). Preferably, CD 34+ cell isolation isperformed first.

Materials and Methods

Placenta donors were recruited from expectant mothers that enrolled inprivate umbilical cord blood banking programs and provided informedconsent permitting the use of the exsanguinated placenta followingrecovery of cord blood for research purposes. These donors alsopermitted use of blinded data generated from the normal processing oftheir umbilical cord blood specimens for cryopreservation. This allowedcomparison between the composition of the collected cord blood and theeffluent perfusate recovered using this experimental method describedbelow. All donor data is confidential.

Following exsanguination of the umbilical cord and placenta, theplacenta was placed in a sterile, insulated container at roomtemperature and delivered to the laboratory within 4 hours of birth.Placentas were discarded if, on inspection, they had evidence ofphysical damage such as fragmentation of the organ or avulsion ofumbilical vessels. Placentas were maintained at room temperature(23+/−2° C.) or refrigerated (4° C.) in sterile containers for 2 to 20hours. Periodically, the placentas were immersed and washed in sterilesaline at 25+/−3° C. to remove any visible surface blood or debris. Theumbilical cod was transected approximately 5 cm from its insertion intothe placenta and the umbilical vessels were cannulated with Teflon orpolypropylene catheters connected to a sterile fluid path allowingbi-directional perfusion of the placenta and recovery of the effluentfluid. The system employed in the present invention enabled all aspectsof conditioning, perfusion and effluent collection to be performed undercontrolled ambient atmospheric conditions as well as real-timemonitoring of intravascular pressure and flow rates, core and perfusatetemperatures and recovered effluent volumes. A range of conditioningprotocols were evaluated over a 24 hour post-partum period and thecellular composition of the effluent fluid was analyzed by flowcytometry, light microscopy and colony forming unit assays.

Placental Conditioning

The placenta was maintained under varying conditions in an attempt tosimulate and sustain a physiologically compatible environment for theproliferation and recruitment of residual cells. The cannula was flushedwith IMDM serum-free medium (GibcoBRL, NY) containing 2 U/ml heparin(EJkins-Sinn, NJ). Perfusion of the placenta continued at a rate of 50mL per minute until approximately 150 mL of perfusate was collected.This volume of perfusate was labeled “early fraction”. Continuedperfusion of the placenta at the same rate resulted in the collection ofa second fraction of approximately 150 mL and was labeled “latefraction”. During the course of the procedure, the placenta was gentlymassaged to aid in the perfusion process and assist in the recovery ofcellular material. Effluent fluid was collected from the perfusioncircuit by both gravity drainage and aspiration through the arterialcannula.

Placentas were obtained from delivery rooms along with cord blood afterobtaining written parental consent, and were processed at roomtemperature within 12 to 24 hours after delivery. Before processing, themembranes were removed and the maternal site washed clean of residualblood. The umbilical vessels were cumulated with catheters made from 20gauge Butterfly needles use for blood sample collection. Placentas werethen perfused with heparinized (2 U/mL) Dulbecco's modified Eagle Medium(H.DMEM) at the rate of 15 mL/minute for 10 minutes and the perfusateswere collected from the maternal sites within one hour and the nucleatedcells counted. The perfusion and collection procedures were repeatedonce or twice until the number of recovered nucleated cells fell below100/microL. The perfusates were pooled and subjected to lightcentrifugation to remove platelets, debris and de-nucleated cellmembranes. The nucleated cells were then isolated by Ficoll-Hypaquedensity gradient contrifugation and after washing, resuspended inH.DMEM. For isolation of the adherent cells, aliquots of 5-10×10⁶ cellswere placed in each of several T-75 flasks and cultured withcommercially available Mesenchymal Stem Cell Growth Medium (MSCGM)obtained from BioWhittaker, and placed in a tissue culture incubator(37° C., 5% CO₂). After 10 to 15 days, the non-adherent cells wereremoved by washing with PBS, which was then replaced by MSCGM. Theflasks were examined daily for the presence of various adherent celltypes and in particular, for identification and expansion of clusters offibroblastoid cells.

Cell Recovery and Isolation

Cells are recovered from the perfusates by centrifugation at X 00×g for15 minutes at room temperature. This procedure served to separate cellsfrom contaminating debris and platelets. The cell pellets wereresuspended in IMDM serum-free medium containing 2 U/ml heparin and 2 mMEDTA (GibcoBRL, NY). The total mononuclear cell fraction was isolatedusing Lymphoprep (Nycomed Pharma, Oslo, Norway) according to themanufacturer's recommended procedure and the mononuclear cell fractionwas resuspended. Cells were counted using a hemocytometer. Viability wasevaluated by trypan blue exclusion. Isolation of mesenchymal cells wasachieved by “differential trypsinization,” using a solution of 0.05%trypsin with 0.2% EDTA (Sigma). Differential trypsinization was possiblebecause fibroblastoid cells detached from plastic surfaces within aboutfive minutes whereas the other adherent populations required more than20-30 minutes incubation. The detached fibroblastoid cells wereharvested following trypsinization and trypsin neutralization, usingTrypsin Neutralyzing Solution (TNS, BioWhitaker). The cells were washedin H.DMEM and resuspended in MSCGM. Flow cytometry was carried out usinga Becton-Dickinson FACSCalibur instrument and FITC and PE labeledmonoclonal antibodies, selected on the basis of known markers for bonemarrow-derived MSC (mesenchymal stem cells), were purchased from B.D.and Caltag laboratories (S. San Francisco, Calif.), and SH2, SH3 and Sh4antibody producing hybridomas were obtained from AM. Cul. andreactivities of the MoAbs in their cultured supernatants were detectedby FITC or PE labeled F(ab)′2 goat anti-mouse antibodies. Lineagedifferentiation was carried out using the commercially availableinduction and maintenance culture media (BioWhittaker), used as permanufacturer's instructions.

Isolation of Placental Stem Cells

Microscopic examination of the adherent cells in the culture flasksrevealed morphologically different cell types. The spindle-shaped cells,the round cells with large nuclei and numerous peri-nuclear smallvacuoles and star-shaped cells with several projections, through one ofwhich the cells were attached to the flask. Although no attempts weremade to further characterize these adherent cells, similar cells wereobserved in the culture of bone marrow, cord and peripheral blood, andtherefore considered to be non-stem cell in nature. The fibroblastoidcells, appearing last as clusters, were candidates for being MSC andwere isolated by differential trypsinization and subcultured insecondary flasks. Phase microscopy of the rounded cells, aftertrypsinization, to be highly granulated; indistinguishable from the bonemarrow-derived MSC produced in the laboratory or purchased fromBioWhittaker. When subcultured, the placental-derived cells, in contrastto their earlier phase, adhered within hours, assumed characteristicfibroblastoid shape, and formed a growth pattern identical to thereference bone marrow-derived MSC. Moreover, during subculturing andrefeeding, the loosely bound mononuclear cells were washed out and thecultures remained homogeneous and devoid of any visiblenon-fibroblastoid cell contaminants.

Flow Cytometry

The expression of CD-34, CD-38, and other stem cell-associated surfacemarkers on early and late fraction purified mononuclear cells wasassessed by flow cytometry. Briefly, cells were washed in PBS and thendouble-stained with anti-CD34 phycoerythrin and anti-CD38 fluoresceinisothiocyanate (Becton Dickinson, Mountain View, Calif.).

Whereas particular embodiments of this invention have been describedherein for purposes of illustration, it will be evident to those personsskilled in the art that numerous variations of the details of thepresent invention may be made without departing from the invention asdefined in the appended claims.

What is claimed is:
 1. A method of collecting a cell populationcomprising, perfusing a post-partum human placenta with a perfusionsolution by passing said perfusion solution into one or both of theumbilical artery and umbilical vein of said placenta; and collectingsaid perfusion solution, wherein said collected perfusion solutioncomprises a cell population comprising a nucleated placental cellpopulation.
 2. The method of claim 1, further comprising isolating thecell population comprising a nucleated cell population from thecollected perfusion solution.
 3. The method of claim 2, wherein the cellpopulation is isolated by centrifugation of the collected perfusionsolution.
 4. The method of claim 2, further comprising removingplatelets from the cell population, thereby producing an isolatednucleated placental cell population.
 5. The method of claim 4, whereinthe nucleated cell population is isolated by density gradientcentrifugation.
 6. The method of claim 4, wherein the collectedperfusion solution consists of perfusion solution that has beencollected from umbilical vessels.
 7. The method of claim 4, wherein thecollected perfusion solution comprises perfusion solution that has beencollected from umbilical vessels and perfusion solution that has beencollected from maternal sites of the placenta.
 8. The method of claim 1,wherein the perfusion solution comprises heparin.
 9. The method of claim1, wherein collecting the perfusion solution comprises collection viagravity drainage.
 10. The method of claim 1, wherein collecting theperfusion solution comprises collection via aspiration.
 11. The methodof claim 1, wherein neither said cell population nor said nucleated cellpopulation are combined with cord blood or hematopoietic stem cells fromcord blood.